Antibody-drug conjugates

ABSTRACT

Disclosed are anti-5T4 antibody drug conjugates and methods for preparing and using the same.

This application claims priority from copending provisional applicationNos. 61/602,349 filed Feb. 23, 2012, 61/593,549 filed Feb. 1, 2012 and61/470,576 filed Apr. 1, 2011 the entire disclosure of which is herebyincorporated by reference.

FIELD

The present invention generally relates to anti-5T4 antibody-drugconjugates for the treatment of cancer.

BACKGROUND

Antibody-drug conjugates (ADCs) combine the binding specificity ofmonoclonal antibodies with the potency of chemotherapeutic agents. Thetechnology associated with the development of monoclonal antibodies totumor associated target molecules, the use of more effective cytotoxicagents, and the design of chemical linkers to covalently bind thesecomponents, has progressed rapidly in recent years (Ducry L., et al.Bioconjugate Chemistry, 21:5-13, 2010).

Promising ADCs such as SGN-75 (U.S.2009/148942) and trastuzumab-DM1(U.S.2009/0226465) are currently in clinical trials. However, as othertumor associated antigens are considered for targets, numerouschallenges remain. Each monoclonal antibody must be characterizedseparately, an appropriate linker designed, and a suitable cytotoxicagent identified that retains its potency upon delivery to tumor cells.One must consider the antigen density on the cancer target and whethernormal tissues express the target antigen. Other considerations includewhether the entire ADC is internalized upon binding the target; whethera cytostatic or cytotoxic drug is preferable when considering possiblenormal tissue exposure and/or the type and stage of the cancer beingtreated; and, whether the linker connecting the antibody to the drugpayload is a cleavable or a non-cleavable linkage. Furthermore, theantibody to drug moiety conjugation ratio must be sufficient withoutcompromising the binding activity of the antibody and/or the potency ofthe drug. It is evident that ADCs are complex biologics and thechallenges to develop an effective ADC remain significant.

The human 5T4 tumor associated antigen is the target antigen of thepresent invention. It has recently been shown that the 5T4 antigen isexpressed in high levels on certain highly tumorigenic cells, alsocalled tumor-initiating cells (WO2010/111659). Tumor-initiating cellsshow resistance to standard therapies and are believed to be responsiblefor tumor recurrence and metastasis and therefore present yet anotherobstacle for ADC development.

The novel anti-5T4 ADCs of the present invention overcome the challengesassociated with ADC technology and provide highly specific and potentADCs that bind to tumor cells expressing the 5T4 antigen and deliversufficient cytotoxic drug to the cells, thus providing an innovative andeffective treatment for cancer.

SUMMARY

In one embodiment, an antibody-drug conjugate of the present inventionhas the formula: Ab-(LU-D)p or a pharmaceutically acceptable saltthereof wherein, Ab is an anti-5T4 antibody or antigen binding portionthereof, comprising a heavy chain variable region having a VH CDR1region as shown in SEQ ID NO: 5, a VH CDR2 region as shown in SEQ ID NO:6, and a VH CDR3 region as shown in SEQ ID NO: 7; LU is a linker unitselected from the group consisting of maleimidocaproyl andmaleimidocaproyl-Val-Cit-PABA; p is an integer from about 1 to about 8;and D is a Drug unit selected from the group consisting of MMAE, MMAF,and MMAD.

The present invention further provides anti-5T4 antibody-drug conjugateswherein said anti-5T4 antibody or antigen binding portion thereof,comprises a heavy chain variable region having (a) a VH CDR1 region asshown in SEQ ID NO: 5, (b) a VH CDR2 region as shown in SEQ ID NO: 6,and (c) a VH CDR3 region as shown in SEQ ID NO: 7.

The present invention further provides an anti-5T4 antibody-drugconjugate wherein said anti-5T4 antibody or antigen binding portionthereof, comprises a light chain variable region having (a) a VL CDR1region as shown in SEQ ID NO: 8, (b) a VL CDR2 region as shown in SEQ IDNO: 9, and (c) a VL CDR3 region as shown in SEQ ID NO: 10.

The present invention further provides an anti-5T4 antibody-drugconjugate wherein said anti-5T4 antibody or antigen binding portionthereof, further comprises a heavy chain variable region having (a) a VHCDR1 region as shown in SEQ ID NO: 5, (b) a VH CDR2 region as shown inSEQ ID NO: 6, and (c) a VH CDR3 region as shown in SEQ ID NO: 7 and alight chain variable region having (a) a VL CDR1 region as shown in SEQID NO: 8, (b) a VL CDR2 region as shown in SEQ ID NO: 9, and (c) a VLCDR3 region as shown in SEQ ID NO: 10.

The present invention further provides an anti-5T4 antibody-drugconjugate wherein said anti-5T4 antibody or antigen binding portionthereof, comprises the VH region of SEQ ID NO: 3 and the VL region ofSEQ ID NO: 4.

The present invention further provides an anti-5T4 antibody-drugconjugate wherein said anti-5T4 antibody consists of a heavy chainhaving SEQ ID NO: 1 and a light chain having SEQ ID NO: 2.

The present invention further provides an anti-5T4 antibody-drugconjugate wherein:

-   (a) said anti-5T4 antibody consists of a heavy chain having SEQ ID    NO:1 and a light chain having SEQ ID NO: 2, (b) said LU is    maleimidocaproyl, (c) said Drug is MMAF, and (d) p is an integer of    about 4.

The present invention further provides an anti-5T4 antibody-drugconjugate wherein:

-   (a) said anti-5T4 antibody consists of a heavy chain having SEQ ID    NO:1 and a light chain having SEQ ID NO: 2, (b) said LU is    maleimidocaproyl-Val-Cit-PABA, (c) said Drug is MMAE, and (d) p is    an integer of about 4.

The present invention further provides an anti-5T4 antibody-drugconjugate wherein:

-   (a) said anti-5T4 antibody consists of a heavy chain having SEQ ID    NO:1 and a light chain having SEQ ID NO: 2, (b) said LU is    maleimidocaproyl-Val-Cit-PABA, (c) said Drug is MMAD, and (d) p is    an integer from about 1 to about 8.

The present invention further provides an anti-5T4 antibody-drugconjugate wherein: (a) said anti-5T4 antibody consists of a heavy chainhaving SEQ ID NO:15 and a light chain having SEQ ID NO: 2, (b) said LUis maleimidocaproyl-Val-Cit-PABA, (c) said Drug is MMAE, and (d) p is aninteger of about 1 to about 8.

The present invention provides an anti-5T4 antibody-drug conjugatewherein said antibody recognizes an epitope on human 5T4 antigen whereinsaid epitope comprises amino acid residues 173-258 and 282-361 of theamino acid sequence of SEQ ID NO: 11.

The present invention provides a pharmaceutical composition comprisingan antibody-drug conjugate indicated above and a pharmaceuticallyacceptable carrier.

The present invention further provides a method of treating a5T4-positive cancer in a patient in need thereof, comprisingadministering to said patient an antibody-drug conjugate indicatedabove.

The present invention further provides a method of treating a5T4-positive cancer wherein said cancer is selected from the groupconsisting of carcinomas of the bladder, breast, cervix, colon,endometrium, kidney, lung, esophagus, ovary, prostate, pancreas, liver,skin, stomach, and testes.

More preferably, the present invention provides a method of treating a5T4-positive cancer wherein said cancer is selected from the groupconsisting of colorectal, breast, pancreatic, and non-small cell lungcarcinomas.

The invention further provides an antibody-drug conjugate indicatedabove for use in therapy. The invention further provides the use of anantibody-drug conjugate indicated above for the manufacture of amedicament.

The invention further provides the use indicated above, wherein said useis for the treatment of a 5T4-positive cancer and wherein said cancer isselected from the group consisting of carcinomas of the bladder, breast,cervix, endometrium, kidney, lung, esophagus, ovary, prostate, pancreas,skin, stomach, and testes.

More preferably, the invention further provides the use indicated above,wherein said use is for the treatment of a 5T4-positive cancer whereinsaid cancer is selected from the group consisting of colorectal, breast,pancreatic, and non-small cell lung carcinomas.

The invention further provides a nucleic acid that encodes an anti-5T4antibody, a vector comprising said nucleic acid, and a host cellcomprising said vector.

The invention further provides a process for producing an anti-5T4antibody comprising cultivating the host cell comprising the abovementioned vector and recovering the antibody from the cell culture.

The invention further provides a process for producing an anti-5T4antibody-drug conjugate comprising: (a) taking the antibody recoveredfrom the cell culture, (b) chemically linking said antibody via a linkerunit selected from the group consisting of maleimidocaproyl ormaleimidocaproyl-Val-Cit to a Drug unit selected from the groupconsisting of MMAE, MMAD, or MMAF, and (c) purifying the antibody-drugconjugate.

DETAILED DESCRIPTION

The present invention provides anti-5T4 antibody-drug conjugates for thetreatment of cancer. In order that the present invention is more readilyunderstood, certain terms are first defined.

All amino acid abbreviations used in this disclosure are those acceptedby the United States Patent and Trademark Office as set forth in 37C.F.R. §1.822 (B)(I).

5T4 refers to the 5T4 oncofetal antigen, a 72 kDa highly glycosylatedtransmenbrance glycoprotein comprising a 42 kDa non-glycosylated core(see U.S. Pat. No. 5,869,053). Human 5T4 is expressed in numerous cancertypes, including carcinomas of the bladder, breast, cervix, colon,endometrium, kidney, lung, esophagus, ovary, prostate, pancreas, liver,skin, stomach, and testes. Highly tumorigenic cells, also called cancerstem cells or tumor-initiating cells have been shown to have high levelsof 5T4 expression (W02010/111659). Anti-5T4 antibodies of the inventioninclude antibodies that specifically bind the human 5T4 antigen (seeU.S. 2007/0231333).

An “antibody” is an immunoglobulin molecule capable of specific bindingto a target, such as a carbohydrate, polynucleotide, lipid, polypeptide,etc., through at least one antigen recognition site, located in thevariable region of the immunoglobulin molecule. As used herein, the term“antibody” encompasses not only intact polyclonal or monoclonalantibodies, but also any antigen binding fragment (i.e.,“antigen-binding portion”) or single chain thereof, fusion proteinscomprising an antibody, and any other modified configuration of theimmunoglobulin molecule that comprises an antigen recognition siteincluding, for example without limitation, Fab, Fab′, F(ab′)₂, an Fdfragment consisting of the VH and CH1 domains, an Fv fragment consistingof the VL and VH domains of a single arm of an antibody, an isolatedcomplementarity determining region (CDR), scFv, single domain antibodies(e.g., shark and camelid antibodies), maxibodies, minibodies,intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv.

An antibody includes an antibody of any class, such as IgG, IgA, or IgM(or sub-class thereof), and the antibody need not be of any particularclass. Depending on the antibody amino acid sequence of the constantregion of its heavy chains, immunoglobulins can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. Theheavy-chain constant regions that correspond to the different classes ofimmunoglobulins are called alpha, delta, epsilon, gamma, and mu,respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.

A “variable region” of an antibody refers to the variable region of theantibody light chain or the variable region of the antibody heavy chain,either alone or in combination. As known in the art, the variableregions of the heavy and light chain each consist of four frameworkregions (FRs) connected by three complementarity determining regions(CDRs) also known as hypervariable regions, contribute to the formationof the antigen binding site of antibodies. If variants of a subjectvariable region are desired, particularly with substitution in aminoacid residues outside of a CDR region (i.e., in the framework region),appropriate amino acid substitution, preferably, conservative amino acidsubstitution, can be identified by comparing the subject variable regionto the variable regions of other antibodies which contain CDR1 and CDR2sequences in the same canonincal class as the subject variable region(Chothia and Lesk, J Mol Biol 196 (4): 901-917, 1987). When choosing FRto flank subject CDRs, e.g., when humanizing or optimizing an antibody,FRs from antibodies which contain CDR1 and CDR2 sequences in the samecanonical class are preferred.

A “CDR” of a variable domain are amino acid residues within the variableregion that are identified in accordance with the definitions of theKabat, Chothia, the cumulation of both Kabat and Chothia, AbM, contact,and/or conformational definitions or any method of CDR determinationwell known in the art. Antibody CDRs may be identified as thehypervariable regions originally defined by Kabat et al. See, e.g.,Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5thed., Public Health Service, NIH, Washington, D.C. The positions of theCDRs may also be identified as the structural loop structures originallydescribed by Chothia and others. See, e.g., Chothia et al., 1989, Nature342:877-883. Other approaches to CDR identification include the “AbMdefinition,” which is a compromise between Kabat and Chothia and isderived using Oxford Molecular's AbM antibody modeling software (nowAccelrys®), or the “contact definition” of CDRs based on observedantigen contacts, set forth in MacCallum et al., 1996, J. Mol. Biol.,262:732-745. In another approach, referred to herein as the“conformational definition” of CDRs, the positions of the CDRs may beidentified as the residues that make enthalpic contributions to antigenbinding. See, e.g., Makabe et al., 2008, Journal of BiologicalChemistry, 283:1156-1166. Still other CDR boundary definitions may notstrictly follow one of the above approaches, but will nonethelessoverlap with at least a portion of the Kabat CDRs, although they may beshortened or lengthened in light of prediction or experimental findingsthat particular residues or groups of residues or even entire CDRs donot significantly impact antigen binding. As used herein, a CDR mayrefer to CDRs defined by any approach known in the art, includingcombinations of approaches. The methods used herein may utilize CDRsdefined according to any of these approaches. For any given embodimentcontaining more than one CDR, the CDRs may be defined in accordance withany of Kabat, Chothia, extended, AbM, contact, and/or conformationaldefinitions.

The term “monoclonal antibody” (Mab) refers to an antibody that isderived from a single copy or clone, including e.g., any eukaryotic,prokaryotic, or phage clone, and not the method by which it is produced.Preferably, a monoclonal antibody of the invention exists in ahomogeneous or substantially homogeneous population.

“Humanized” antibody refers to forms of non-human (e.g. murine)antibodies that are chimeric immunoglobulins, immunoglobulin chains, orfragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) that contain minimalsequence derived from non-human immunoglobulin. Preferably, humanizedantibodies are human immunoglobulins (recipient antibody) in whichresidues from a complementary determining region (CDR) of the recipientare replaced by residues from a CDR of a non-human species (donorantibody) such as mouse, rat, or rabbit having the desired specificity,affinity, and capacity.

The term “chimeric antibody” is intended to refer to antibodies in whichthe variable region sequences are derived from one species and theconstant region sequences are derived from another species, such as anantibody in which the variable region sequences are derived from a mouseantibody and the constant region sequences are derived from a humanantibody.

Antibodies of the invention can be produced using techniques well knownin the art, e.g., recombinant technologies, phage display technologies,synthetic technologies or combinations of such technologies or othertechnologies readily known in the art (see, for example, Jayasena, S.D., Clin. Chem., 45: 1628-50 (1999) and Fellouse, F. A., et al, J. Mol.Biol., 373 (4):924-40 (2007)).

Tables 1 and 2 below depict preferred CDRs for the antibodies of thepresent invention.

TABLE 1 Anti- body LCDR1 LCDR2 LCDR3 A1 KASQSVSNDVA FATNRYT QQDYSSPWTSEQ ID NO: 8 SEQ ID NO: 9 SEQ ID NO: 10 A3 KASQDVDTAVA WASTRLT QQYSSYPYTSEQ ID NO: 17 SEQ ID NO: 18 SEQ ID NO: 19

TABLE 2 Anti- body HCDR1 HCDR2 HCDR3 A1 NFGMN WINTNTGEPRYAEEFKGDWDGAYFFDY SEQ ID NO: 5 SEQ ID NO: 6 SEQ ID NO: 7 A1- GYTFTNFGMNWINTNTGEPRYAEEFKG DWDGAYFFDY IgG4 SEQ ID NO: 14 SEQ ID NO: 6SEQ ID NO: 7 A3 TYAMN RIRSKSNNYATYYADSVKD QWDYDVRAMNY SEQ ID NO: 22SEQ ID NO: 23 SEQ ID NO: 24

The present invention includes an antibody or antigen binding portionthereof, that comprises:

a) a light chain variable region comprising:

-   -   i) a LCDR1 having an amino acid sequence selected from the group        consisting of SEQ ID NOs: 8 and 17;    -   ii) a LCDR2 having an amino acid sequence selected from the        group consisting of SEQ ID NOs: 9 and 18; and    -   iii) a LCDR3 having an amino acid sequence selected from the        group consisting of SEQ ID NOs: 10 and 19; and

b) a heavy chain variable region comprising:

-   -   i) a HCDR1 having an amino acid sequence selected from the group        consisting of SEQ ID NOs: 5 and 22;    -   ii) a HCDR2 having an amino acid sequence selected from the        group consisting of SEQ ID NOs: 6 and 23; and p1 iii) a LCDR1        having an amino acid sequence selected from the group consisting        of SEQ ID NOs: 7 and 24.

A preferred antibody or antigen binding portion thereof, of theinvention comprises:

a) a LCVR comprising: a LCDR1 of SEQ ID NO: 8, a LCDR2 of SEQ ID NO: 9,and a LCDR3 of SEQ ID NO: 10; and

b) a HCVR comprising: a HCDR1 of SEQ ID NO: 5, a HCDR2 of SEQ ID NO: 6,and a HCDR3 of SEQ ID NO: 7.

Preferred monoclonal antibodies of the invention are referred to hereinas A1 (a humanized anti-5T4 IgG1 antibody); A1-IgG4 (a humanizedanti-5T4 IgG4 antibody); A3 (a mouse/human chimeric antibody); and A3hu(a humanized anti-5T4 IgG1 antibody). The SEQ ID NOs of the amino acidsequences encoding Mabs A1, A1-IgG4 and A3 are provided in Table 3below:

TABLE 3 Mab LC HC LCVR LCDR1 LCDR2 LCDR3 HCVR HCDR1 HCDR2 HCDR3 A1  2  1 4  8  9 10  3  5  6  7 A1-  2 12  4  8  9 10 13  5  6  7 IgG4 A3  2 1521 22 23 24 16 17 18 19 A3hu 30 25 31 32 33 34 26 27 28 29

The phrases “an antibody recognizing an antigen” and “an antibodyspecific for an antigen” are used interchangeably herein with the term“an antibody which binds specifically to an antigen.”

Anti-5T4 Antibody-Drug conjugate refers to an anti-5T4 antibody orantigen binding portion thereof, as described herein linked to acytotoxic drug moiety (D) via a linker unit molecule (LU).

Linker Unit (LU): LU describes the direct or indirect linkage of theantibody to the drug. Attachment of a linker to a mAb can beaccomplished in a variety of ways, such as through surface lysines,reductive-coupling to oxidized carbohydrates, and through cysteineresidues liberated by reducing interchain disulfide linkages. A varietyof ADC linkage systems are known in the art, including hydrazone-,disulfide- and peptide-based linkages.

Drug (D): A drug is any substance having biological or detectableactivity, for example, therapeutic agents, detectable labels, bindingagents, etc., and prodrugs, which are metabolized to an active agent invivo. The terms drug and payload are used interchangeably. In someembodiments, the Drug is an auristatin, such as auristatin E (also knownin the art as a derivative of dolastatin-10) or a derivative thereof.The auristatin can be, for example, an ester formed between auristatin Eand a keto acid. For example, auristatin E can be reacted withparaacetyl benzoic acid or benzoylvaleric acid to produce AEB and AEVB,respectively. Other typical auristatins include AFP, MMAF, and MMAE. Thesynthesis and structure of exemplary auristatins are described in U.S.Pat. Nos. 6,884,869, 7,098,308, 7,256,257, 7,423,116, 7,498,298 and7,745,394, each of which is incorporated by reference herein in itsentirety and for all purposes.

Auristatins have been shown to interfere with microtubule dynamics andnuclear and cellular division and have anticancer activity. Auristatinsof the present invention bind tubulin and can exert a cytotoxic orcytostatic effect on a 5T4 expressing cell or cell line. There are anumber of different assays, known in the art, that can be used fordetermining whether an auristatin or resultant antibody-drug conjugateexerts a cytostatic or cytotoxic effect on a desired cell or cell line.Methods for determining whether a compound binds tubulin are known inthe art. See, for example, Muller et al., Anal. Chem 2006, 78,4390-4397; Hamel et al., Molecular Pharmacology, 1995 47: 965-976; andHamel et al., The Journal of Biological Chemistry, 1990 265:28,17141-17149.

Examples of drugs or payloads are selected from the group consisting ofDM1 (maytansine, N2′-deacetyl-N2′-(3-mercapto-1-oxopropyl)- orN2′-deacetyl-N2′-(3-mercapto-1-oxopropyl)-maytansine), mc-MMAD(6-maleimidocaproyl-monomethylauristatin-D orN-methyl-L-valyl-N-[(1S,2R)-2-methoxy-4-[(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-[[(1S)-2-phenyl-1-(2-thiazolyl)ethyl]amino]propyl]-1-pyrrolidinyl]-1-[(1S)-1-methylpropyl]-4-oxobutyl]-N-methyl-(9Cl)-L-valinamide),mc-MMAF (maleimidocaproyl-monomethylauristatin F orN-[6-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-1-oxohexyl]-N-methyl-L-valyl-L-valyl-(3R,4S,5S)-3-methoxy-5-methyl-4-(methylamino)heptanoyl-(αR,βR,2S)-β-(3-methoxy-α-methyl-2-pyrrolidinepropanoyl-L-phenylalanine)and mc-Val-Cit-PABA-MMAE(6-maleimidocaproyl-ValcCit-(p-aminobenzyloxycarbonyl)-monomethylauristatinE orN-[[[4-[[N-[6-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-1-oxohexyl]-L-valyl-N5-(aminocarbonyl)-L-ornithyl]amino]phenyl]methoxy]carbonyl]-N-methyl-L-valyl-N-[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenylethyl]amino]-1-methoxy-2-methyl-3-oxopropyl]-1-pyrrolidinyl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl]-N-methyl-L-valinamide).DM1 is a derivative of the tubulin inhibitor maytansine while MMAD,MMAE, and MMAF are auristatin derivatives. The preferred payloads of thepresent invention are selected from the group consisting of mc-MMAF andmc-Val-Cit-PABA-MMAE.

The term “epitope” refers to that portion of a molecule capable of beingrecognized by and bound by an antibody at one or more of the antibody'santigen-binding regions. Epitopes often consist of a chemically activesurface grouping of molecules such as amino acids or sugar side chainsand have specific three-dimensional structural characteristics as wellas specific charge characteristics. The term “antigenic epitope” as usedherein, is defined as a portion of a polypeptide to which an antibodycan specifically bind as determined by any method well known in the art,for example, by conventional immunoassays. A “nonlinear epitope” or“conformational epitope” comprises noncontiguous polypeptides (or aminoacids) within the antigenic protein to which an antibody specific to theepitope binds.

The term “binding affinity (K_(D))” as used herein, is intended to referto the dissociation rate of a particular antigen-antibody interaction.The K_(D) is the ratio of the rate of dissociation, also called the“off-rate (k_(off))”, to the association rate, or “on-rate (k_(on))”.Thus, K_(D) equals k_(off)/k_(on) and is expressed as a molarconcentration (M). It follows that the smaller the K_(D), the strongerthe affinity of binding. Therefore, a K_(D) of 1 μM indicates weakbinding affinity compared to a K_(D) of nM. K_(D) values for antibodiescan be determined using methods well established in the art. One methodfor determining the K_(D) of an antibody is by using surface plasmonresonance (SPR), typically using a biosensor system such as a Biacore®system.

The term “specifically binds” as used herein in reference to the bindingbetween an antibody and a 5T4 antigen and the antibody binds the 5T4antigen with a K_(D) less than about 30 nM as determined by SPR at 25°C.

Pharmaceutically acceptable salt as used herein refers topharmaceutically acceptable organic or inorganic salts of a molecule ormacromolecule.

The term “potency” is a measurement of biological activity and may bedesignated as I₅₀, or effective concentration of antibody needed toinhibit 50% of growth of a 5T4 positive cell line as described inExample 3. Alternatively, potency may refer to anti-tumor activity asdetermined in an in vivo tumor xenograph model as shown in Example 4.

The terms “polynucleotide” or “nucleic acid molecule”, as used herein,are intended to include DNA molecules and RNA molecules. A nucleic acidmolecule may be single-stranded or double-stranded, but preferably isdouble-stranded DNA.

The polynucleotides that encode the antibodies of the present inventionmay include the following: only the coding sequence for the variant, thecoding sequence for the variant and additional coding sequences such asa functional polypeptide, or a signal or secretory sequence or apro-protein sequence; the coding sequence for the antibody andnon-coding sequence, such as introns or non-coding sequence 5′ and/or 3′of the coding sequence for the antibody. The term ‘polynucleotideencoding an antibody” encompasses a polynucleotide which includesadditional coding sequence for the variant but also a polynucleotidewhich includes additional coding and/or non-coding sequence. It is knownin the art that a polynucleotide sequence that is optimized for aspecific host cell/expression system can readily be obtained from theamino acid sequence of the desired protein (see GENEART® AG, Regensburg,Germany).

The polynucleotides encoding the antibodies of the present inventionwill typically include an expression control polynucleotide sequenceoperably linked to the antibody coding sequences, includingnaturally-associated or heterologous promoter regions known in the art.Preferably, the expression control sequences will be eukaryotic promotersystems in vectors capable of transforming or transfecting eukaryotichost cells, but control sequences for prokaryotic hosts may also beused. Once the vector has been incorporated into the appropriate hostcell line, the host cell is propagated under conditions suitable forexpressing the nucleotide sequences, and, as desired, for the collectionand purification of the antibodies. Preferred eukaryotic cell linesinclude the CHO cell lines, various COS cell lines, HeLa cells, myelomacell lines, transformed B-cells, or human embryonic kidney cell lines.The most preferred host cell is a CHO cell line.

The present invention encompasses antibodies or antigen-binding portionsthereof that bind to a specific epitope on the 5T4 antigen. The epitopeidentified is a nonlinear or conformational epitope comprising a firstcontact with the human 5T4 antigen (SEQ ID NO: 11) between amino acidresidues 173 and 252 and comprising a second contact between amino acidresidues 276 and 355 (see Example 7). Thus, the CDRs and heavy and lightchain variable regions described herein are used to make full-lengthantibodies as well as functional fragments and analogs that maintain thebinding affinity of the protein employing the CDRs specific for theabove mentioned epitope of the 5T4 antigen.

The binding affinity of antibodies of the present invention isdetermined using SPR (Example 6). In these experiments the 5T4 antigensare immobilized at low densities onto a BIAcore® chip and antibodies areflowed past. Build up of mass at the surface of the chip is measured.This analytical method allows the determination in real time of both onand off rates to obtain affinity (K_(D)) for binding. The humanizedantibodies of the present invention have a K_(D) of between about 0.30and about 30 nM; about 0.30 and about 20 nM; about 0.30 and about 10 nM;about 0.5 and about 7 nM; about 1.0 and about 5 nM; and about 1.0 andabout 3 nM.

Conjugation of Drugs to an Antibody

The drug has, or is modified to include, a group reactive with aconjugation point on the antibody. For example, a drug can be attachedby alkylation (e.g., at the epsilon-amino group lysines or theN-terminus of antibodies), reductive amination of oxidized carbohydrate,transesterification between hydroxyl and carboxyl groups, amidation atamino groups or carboxyl groups, and conjugation to thiols. In someembodiments, the number of drug moieties, p, conjugated per antibodymolecule ranges from an average of 1 to 8; 1 to 7, 1 to 6, 1 to 5, 1 to4, 1 to 3, or 1 to 2. In some embodiments, p ranges from an average of 2to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4 or 2 to 3. In other embodiments, pis an average of 1, 2, 3, 4, 5, 6, 7 or 8. In some embodiments, p rangesfrom an average of about 1 to about 8; about 1 to about 7, about 1 toabout 6, about 1 to about 5, about 1 to about 4, about 1 to about 3, orabout 1 to about 2. In some embodiments, p ranges from about 2 to about8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 2to about 4 or about 2 to about 3. For examples of chemistries that canbe used for conjugation, see, e.g., Current Protocols in Protein Science(John Wiley & Sons, Inc.), Chapter 15 (Chemical Modifications ofProteins) (the disclosure of which is incorporated by reference hereinin its entirety.)

For example, when chemical activation of the protein results information of free thiol groups, the protein may be conjugated with asulfhydryl reactive agent. In one aspect, the agent is one which issubstantially specific for free thiol groups. Such agents include, forexample, malemide, haloacetamides (e.g., iodo, bromo or chloro),haloesters (e.g., iodo, bromo or chloro), halomethyl ketones (e.g.,iodo, bromo or chloro), benzylic halides (e.g., iodide, bromide orchloride), vinyl sulfone and pyridylthio.

Linkers

The drug can be linked to an antibody by a linker. Suitable linkersinclude, for example, cleavable and non-cleavable linkers. A cleavablelinker is typically susceptible to cleavage under intracellularconditions. Suitable cleavable linkers include, for example, a peptidelinker cleavable by an intracellular protease, such as lysosomalprotease or an endosomal protease. In exemplary embodiments, the linkercan be a dipeptide linker, such as a valine-citrulline (val-cit), aphenylalanine-lysine (phe-lys) linker, ormaleimidocapronic-valine-citruline-p-aminobenzyloxycarbonyl(mc-Val-Cit-PABA) linker. Another linker isSulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (smcc).Sulfo-smcc conjugation occurs via a maleimide group which reacts withsulfhydryls (thiols, -SH), while its Sulfo-NHS ester is reactive towardprimary amines (as found in Lysine and the protein or peptideN-terminus). Yet another linker is maleimidocaproyl (mc). Other suitablelinkers include linkers hydrolyzable at a specific pH or a pH range,such as a hydrazone linker. Additional suitable cleavable linkersinclude disulfide linkers. The linker may be covalently bound to theantibody to such an extent that the antibody must be degradedintracellularly in order for the drug to be released e.g. the mc linkerand the like.

A linker can include a group for linkage to the antibody. For example,linker can include an amino, hydroxyl, carboxyl or sulfhydryl reactivegroups (e.g., malemide, haloacetamides (e.g., iodo, bromo or chloro),haloesters (e.g., iodo, bromo or chloro), halomethyl ketones (e.g.,iodo, bromo or chloro), benzylic halides (e.g., iodide, bromide orchloride), vinyl sulfone and pyridylthio). See generally Wong, Chemistryof Protein Conjugation and Cross-linking; CRC Press, Inc., Boca Raton,1991.

Immunotherapy

For immunotherapy, an antibody can be conjugated to a suitable drug,such as a cytotoxic or cytostatic agent, an immunosuppressive agent, aradioisotope, a toxin, or the like. The conjugate can be used forinhibiting the multiplication of a tumor cell or cancer cell, causingapoptosis in a tumor or cancer cell, or for treating cancer in apatient. The conjugate can be used accordingly in a variety of settingsfor the treatment of animal cancers. The conjugate can be used todeliver a drug to a tumor cell or cancer cell. Without being bound bytheory, in some embodiments, the conjugate binds to or associates with acancer-cell or a tumor-associated antigen, and the conjugate and/or drugcan be taken up inside a tumor cell or cancer cell throughreceptor-mediated endocytosis. The antigen can be attached to a tumorcell or cancer cell or can be an extracellular matrix protein associatedwith the tumor cell or cancer cell. Once inside the cell, one or morespecific peptide sequences within the conjugate (e.g., in a linker) arehydrolytically cleaved by one or more tumor-cell orcancer-cell-associated proteases, resulting in release of the drug. Thereleased drug is then free to migrate within the cell and inducecytotoxic or cytostatic or other activities. In some embodiments, thedrug is cleaved from the antibody outside the tumor cell or cancer cell,and the drug subsequently penetrates the cell, or acts at the cellsurface.

Therapy for Cancer

As discussed above, cancers, including, but not limited to, a tumor,metastasis, or other disease or disorder characterized by uncontrolledcell growth, can be treated or prevented by administration of aprotein-drug conjugate.

In other embodiments, methods for treating or preventing cancer areprovided, including administering to a patient in need thereof aneffective amount of a conjugate and a chemotherapeutic agent. In someembodiments, the chemotherapeutic agent is that with which treatment ofthe cancer has not been found to be refractory. In some embodiments, thechemotherapeutic agent is that with which the treatment of cancer hasbeen found to be refractory. The conjugate can be administered to apatient that has also undergone a treatment, such as surgery fortreatment for the cancer. In another embodiment, the additional methodof treatment is radiation therapy.

Multi-Drug Therapy for Cancer

Methods for treating cancer include administering to a patient in needthereof an effective amount of an antibody-drug conjugate and anothertherapeutic agent that is an anti-cancer agent. Suitable anticanceragents include, but are not limited to, methotrexate, taxol,L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine,cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin,mitomycin, dacarbazine, procarbizine, topotecan, nitrogen mustards,cytoxan, etoposide, 5-fluorouracil, BCNU, irinotecan, camptothecins,bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin,plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine,vinorelbine, paclitaxel, calicheamicin, and docetaxel.

The ADCs of the present invention can be in the form of a pharmaceuticalcomposition for administration that are formulated to be appropriate forthe selected mode of administration, and pharmaceutically acceptablediluent or excipients, such as buffers, surfactants, preservatives,solubilizing agents, isotonicity agents, stabilizing agents, carriers,and the like. Remington's Pharmaceutical Sciences, Mack Publishing Co.,Easton Pa., 18^(th) ed., 1995, incorporated herein by reference,provides a compendium of formulation techniques as are generally knownto practitioners.

These pharmaceutical compositions may be administered by any means knownin the art that achieve the generally intended purpose to treat cancer.The preferred route of administration is parenteral, defined herein asreferring to modes of administration that include but not limited tointravenous, intramuscular, intraperitoneal, subcutaneous, andintraarticular injection and infusion. The dosage administered will bedependent upon the age, health, and weight of the recipient, kind ofconcurrent treatment, if any, frequency of treatment, and the nature ofthe effect desired.

Compositions within the scope of the invention include all compositionswherein an ADC is present in an amount that is effective to achieve thedesired medical effect for treating cancer. While individual needs mayvary from one patient to another, the determination of the optimalranges of effective amounts of all of the components is within theability of the clinician of ordinary skill.

EXAMPLE 1 Preparation of an Anti-5T4 ADC

5T4-A1 antibody drug conjugate (ADC) is prepared via partial reductionof the mAb with tris(2-carboxyethyl)phosphine (TCEP) followed byreaction of reduced Cys residues with the desired maleimide terminatedlinker-payload. In particular, 5T4-A1 mAb is partially reduced viaaddition of 2.8 molar excess of tris(2-carboxyethyl)phosphine (TCEP) in100 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidbuffer), pH 7.0 and 1 mM diethylenetriaminepentaacetic acid (DTPA) for 2h at 37° C. The desired linker-payload is then added to the reactionmixture at a linker-payload/mAb-thiol molar ratio of 5.5(maleimidocapronic-monomethylauristatin F [mc-MMAF]) or 8(maleimidocapronic-valine-citruline-p-aminobenzyloxycarbonyl-monomethylauristatinE [mc-Val-Cit-PABA-MMAE]) and reacted for an additional 1 h at 25° C. inthe presence of 15% v/v of dimethylacetamide (DMA). After the 1 hincubation period, N-ethylmaleimide (4.5 fold excess for mc-MMAF and 2fold excess for mc-Val-Cit-PABA-MMAE) is added to cap the unreactedthiols and is allowed to react for 15 minutes followed by addition of 6fold excess L-Cys to quench any unreacted linker-payload. The reactionmixture is dialyzed overnight at 4° C. in phosphate buffered saline(PBS), pH 7.4, and purified via SEC (AKTA explorer, Superdex200 10/30 GLcolumn). The ADC is further characterized via size exclusionchromatography (SEC) for purity, hydrophobic interaction chromatography(HIC), and liquid chromatography electrospray ionisation tandem massspectrometry (LC-ESI MS) to calculate loading, and the concentration isdetermined via UV spectrophotometer.

EXAMPLE 2 Binding Studies

Cells expressing the 5T4 antigen, and the negative control Raji cells,are plated at a density of 500,000 cells/well on non-tissue culturetreated 96 well plates and kept on ice. Dilutions of the A1 and A1-IgG4antibodies or A1-mcMMAF ADC are made in 3% bovine serum albumin BSA inDulbecco's phosphate buffered saline (DPBS) and added to the plate at afinal concentration of 10 μg/mL. The plates are then incubated on icefor 1 hour followed by 2 washes. The secondary antibody, PE(phycoerythrin) conjugated Goat Anti-Human IgG Fc is added to the wells.After 30 minutes of incubation at 4 C, the mean fluorescence intensityis then measured using a flow cytometer.

The data in Table 4 indicates that the A1 antibody binds a diverse panelof 5T4 positive cell lines. The data in Table 5 indicates that similarbinding on several different cell lines is observed with the A1 andA1-IgG4 antibodies as well as the A1-mcMMAF ADC.

TABLE 4 A1 antibody Human Cell Lines Mean Fluorescent IntensityMDAMB435/5T4 (melanoma) 15000 MDAMB468 (breast) 3000 MDAMD361-DYT2(breast) 4500 NCI-H157 (lung) 4100 A431 (epithelial) 2000 Caki (kidney)2500 PC3mm2 (prostate) 4500 PC14PE6 (lung) 3200 Panc1 (pancreatic) 3500BxPC3 (pancreatic 1000 Su8686 (pancreatic) 3700 H1975 160037622A-(Primary Lung cancer cells) 10600 Raji (negative control) <100

TABLE 5 Mean Fluorescent Intensity A1- A1- A1- Cell Lines A1 mcMMAF IGG4IGG4-CM IgG control MDAMD361- 7000 6900 5500 4800 <100 DYT2 A431 39003400 2400 2000 <100 MDAMB468 3500 2800 2500 1800 <100 PC3mm2 3400 27002100 1500 <100 Raji <200 <200 <200 <100 <100

EXAMPLE 3 Cytotoxicity Assay

Cell lines expressing 5T4, and the negative control Raji cell line, arecultured with increasing concentrations of ADC. After four days,viability of each culture is assessed. IC₅₀ values are calculated bylogistic non-linear regression and are presented as ng Ab/mL. A1-mcMMAF,A1-vcMMAE, A3-mcMMAF and A3-mcMMAE are shown to inhibit the growth of5T4 expressing cell lines (MDAMB435/5T4, MDAMB468, and MDAMB361DYT2),while being inactive on 5T4 negative cells (Raji), Table 6.

TABLE 6 IC₅₀ (ng Ab/ml) MDAMB435/ Raji ADC 5T4 MDAMB361DYT2 MDAMB468(5T4-) A1- 1.3 104.2 534.2 >45,000 mcMMAF A1- 6.8 157.7 7667 >45,000vcMMAE A3- 0.3 31.0 27.7 >45,000 mcMMAF A3- 3.5 86.8 160.6 >45,000vcMMAE Non- 21258 ~50,000 73059 >45,000 binding Ab- mcMMAF Non- 797927650 23819 >45,000 binding Ab- vcMMAE

Additionally, 5T4+primary lung tumor 37622a cells are isolated and grownin culture. Cells are cultured with increasing concentrations of ADC.Ten days later, viability of each culture is assessed using the MTSmethod. IC₅₀ values were calculated by logistic non-linear regressionand are presented as ng Ab/ml. A1-mcMMAF, A1-vcMMAE, A3-mcMMAF, andA3-vcMMAE inhibit the growth of the primary lung tumor cells, Table 7.

TABLE 7 37622a primary lung ADC IC₅₀ (ng Ab/ml) A1-mcMMAF 504.1A1-vcMMAE 443.1 A3-mcMMAF 77.2 A3-vcMMAE 78.1 Nonbinding Ab- >45,000mcMMAF

EXAMPLE 4 Subcutaneous Xenograft Model

Female, athymic (nude) mice (or another strain of immunosupressed mice)are injected s.c. with MDAMB435/5T4, MDAMB361DYT2, or H1975 tumor cells.Mice with staged tumors, approximately 0.1 to 0.3 g (n=6 to 10mice/treatment group) are administered intravenously Q4D×4 with normalsaline (vehicle), A1-mcMMAF, A1-vcMMAE, A1-mcMMAD, A1-smccDM1,A3-mcMMAF, A3-vcMMAE, or a nonbinding control antibody conjugated toeither mcMMAF or vcMMAE, at the dose of 3 mg Ab/kg. All ADCs are dosedbased on Ab content. Tumors are measured at least once a week and theirsize (mm²±SEM) is calculated as mm²=0.5× (tumor width²)×(tumor length).

The data in Table 8 indicates that A1-mcMMAF, A1-vcMMAE, A1-vcMMAD,A3-mcMMAF, and A3-vcMMAE inhibit the growth of MDAMB435/5T4 xenograftswhile A1-mcMMAD and A1-smccDM1 were not active in this model.

The data in Table 9 indicates that A1-mcMMAF, A1-vcMMAE, A1-vcMMAD,A1-smccDM1, A3-mcMMAF, and A3-vcMMAE inhibit the growth of MDAMB361DYT2xenografts while A1-mcMMAD was not active in this model.

The data in Table 10 indicates that A1-mcMMAF, A1-vcMMAE, A1-vcMMAD,A3-mcMMAF, and A3-vcMMAE inhibit the growth of H1975 xenografts whileA1-mcMMAD and A1-smccDM1 were not active in this model.

TABLE 8 Compound MDAMB435/5T4 xenografts (3 mg/kg Tumor volume (mm³, x ±sem) Q4dx4) Day 0 Day 17 Day 42 Day 65 Day 85 Vehicle 169 ± 8  531 ± 731255 ± 190 GT GT A1 168 v 15  53 ± 12  67 ± 56 174 ± 119 364 ± mcMMAF278 A1 168 ± 8   4 ± 4  10 ± 10 91 ± 91 200 ± vcMMAE 200 A1 168 ± 12 390 ± 112 GT GT GT mcMMAD A1 174 ± 10 429 ± 62 1255 ± 227 1781 ± GTsmccDM1 388 A1 169 ± 12 17 ± 7  0 ± 0 0 ± 0 0 ± 0 vcMMAD A3 174 ± 12 105± 27  216 ± 143 448 ± 220 GT mcMMAF A3 172 ± 13  0 ± 0  0 ± 0 0 ± 0 0 ±0 vcMMAE Nonbinding 170 ± 11 100 ± 15  314 ± 121 838 ± 381 GT Ab mcMMAFNonbinding 172 ± 11 168 ± 53  461 ± 178 GT GT Ab vcMMAE GT = groupterminated due to large tumor size

TABLE 9 Compound MDAMB361DYT2 xenografts (3 mg/kg Tumor volume (mm³, x ±sem) Q4dx4) Day 0 Day 19 Day 47 Day 90 Day 131 Vehicle 353 ± 10 363 ± 58558 ± 149 1117 ±  GT 348 A1 348 ± 14  76 ± 32 0 ± 0 7 ± 7 11 ± 11 mcMMAFA1 356 ± 11 86 ± 8 0 ± 0 9 ± 9 34 ± 27 vcMMAE A1 352 ± 26 130 ± 15 0 ± 00 ± 0 0 ± 0 vcMMAD A3 342 ± 23 128 ± 10 79 ± 30 105 ± 353 ± 234 mcMMAF 49 A3 354 ± 21 111 ± 20 21 ± 21 72 ± 72 155 ± 155 vcMMAE A1 347 ± 15380 ± 66 775 ± 199 GT GT mcMMAD A1 352 ± 26 130 ± 15 0 ± 0 0 ± 0 0 ± 0vcMMAD A1 353 ± 25 123 ± 9  51 ± 25  98 ± 330 ± 146 smccDM1  41Nonbinding 342 ± 38 407 ± 93 869 ± 198 GT GT Ab mcMMAF Nonbinding 344 ±20 303 ± 78 346 ± 185 595 ± GT Ab 362 vcMMAE GT = group terminated dueto large tumor size

TABLE 10 H1975 Xenografts Dose (mg/kg) Tumor volume (mm³, x ± sem)Compound Q4dx4 Day 0 Day 8 Day 15 Day 22 Day 40 Vehicle 423 ± 14 1154 ±136 2229 ± 240 GT GT A1 3 425 ± 14 619 ± 46 519 ± 45 581 ± 79 2840 ± 207mcMMAF A1 3 425 ± 12 702 ± 45 929 ± 90 926 ± 116 GT vcMMAE A1 3 427 ± 18739 ± 59 467 ± 19 240 ± 14 625 ± 317 vcMMAD A3 3 426 ± 10 980 ± 79 1343± 140 1261 ± 203 GT mcMMAF A3 3 431 ± 14 944 ± 52 993 ± 71 GT GT vcMMAEA1 3 427 ± 16 837 ± 69 1468 ± 139 GT GT mcMMAD A1 3 423 ± 18 901 ± 831852 ± 167 GT GT smccDM1 Nonbinding 3 423 ± 16 1026 ± 68 1861 ± 224 GTGT Ab- mcMMAF Nonbinding 3 427 ± 13 1213 ± 67 1959 ± 139 GT GT Ab-vcMMAEGT = group terminated due to large tumor size

Alternatively, nude mice with 37622a primary tumor cell xenograftsestablished subcutaneously are treated iv Q4D×4 with A1-mcMMAF,A1-mcMMAD, A1-vcMMAD, or A3-mcMMAF at the dose of 3 mg Ab/kg and thetumor growth is monitored over the period of 96 days. Table 11demonstrates that A1-mcMMAF, A1-vcMMAD and A3-mcMMAF inhibit the growthof 37622a primary tumor xenografts compared to vehicle control treatedanimals while A1-mcMMAD was not active in this model.

TABLE 11 37622a Primary Tumor Xenografts Tumor volume (mm³, x ± sem)Dose (mg/kg) Compound Q4dx4 Day 1 Day 22 Day 46 Day 68 Day 96 Vehicle111 ± 18 503 ± 155 1174 ± 247 GT GT A1- 3 111 ± 18 67 ± 11 124 ± 47 233± 105 357 ± 150 mcMMAF A1- 3 127 ± 28 376 ± 119 862 ± 377 GT GT mcMMADA1- 3 108 ± 14 52 ± 14 13 ± 5 50 ± 37 160 ± 121 vcMMAD A3- 3 131 ± 28 99± 26 211 ± 128 463 ± 210 GT mcMMAF GT = group terminated due to largetumor size

Unexpectedly, the data in Tables 8-11 show that ADCs with the sameantibody and drug payload but with different linkers had a dissimilarefficacy profile i.e. A1-mcMMAD vs A1-vcMMAD in all four xenograftmodels. In addition, the data show that ADCs with the same antibody andlinker but with different drug payloads also had a different efficacyprofile i.e. A1-mcMMAF vs A1-mcMMAD, in all four xenograft models. Thus,the drug MMAD is effective in all four xenograft models when linked tothe A1 antibody by the vc linker but has no activity in any of thexenograft models tested when linked by the mc linker. In contrast, thedrug MMAF is highly effective in all 4 xenograft models when linked tothe A1 antibody with the mc linker while the chemically related drugMMAD has no activity in all 4 xenograft models when linked to the sameantibody by the same linker.

Yet another unexpected observation is seen with the ADC A1-smccDM1(Tables 8-10). This ADC was very effective against the MDAMB361DYT2xenograft but had essentially no effect against the MDAMB435/5T4 and theH1975 xenografts even though all the xenografts have a high expressionof the 5T4 target antigen. This data illustrates that the effectivenessof the linker-payload could not be predicted even when the same highaffinity antibody is utilized or even when the same ADC is used.

EXAMPLE 5 Antibody Dependent Cell-Mediated Cytotoxicity (ADCC) ADCCAssay

Blood from a healthy volunteer is collected into a BD Vacutainer CPTcell preparation tube with sodium heparin. Human peripheral bloodmononucleocytes (PBMC) are harvested and resuspended in assay buffer(RPMI 1640 supplemented with 10 mM HEPES) at 2.5×10⁷ cells/ml. Targetcells (MDAMB435/5T4 or MDAMB435/neo) are seeded at a density of 1×10⁴cells/well in a 96 well assay plate. A1 antibody or A1-mcMMAF are added,then human PBMC effector cells (5×10⁵) are dispensed into the wells foran effector:target cell ratio (E:T) of 50:1. The assay plate isincubated at 37° C. for 4 hours for ADCC activity. The plate isharvested by adding equal volume of CytoTox-One reagent (Promega). Stopsolution (Promega; 50 ul) is added to each well and lactatedehydrogenase release was quantified by measuring fluorescenceintensity. As a positive control, 2 μl of lysis buffer per well is addedto generate a maximum LDH release (100% cytotoxicity) in control wells.Percent cytotoxicity is calculated using the following equation:

${\% \mspace{14mu} {Specific}\mspace{14mu} {Cytotoxicity}} = {\frac{{experimental} - {{effector}\mspace{14mu} {spontaneous}} - {{target}\mspace{14mu} {spontaneous}}}{{{target}\mspace{14mu} {maximum}} - {{target}\mspace{14mu} {spontaneous}}} \times 100}$

Where “experimental” corresponds to the signal measured in one of theexperimental conditions, “effector spontaneous” corresponds to thesignal measured in the presence of PBMC alone, “target spontaneous”corresponds to the signal measured in the presence of target cellsalone, and “target maximum” corresponds to the signal measured in thepresence of detergent-lysed target cells alone.

The ADCC activity of A1-IgG1 Ab and A1-mcMMAF compared to A1-IgG4 Ab isshown in Table 12. Both the A1 antibody and A1-mcMMAF demonstatecomparable ADCC activity indicating that the ADCC activity of A1-mcMMAFmay contribute to its anti-tumor activity.

TABLE 12 Compound % Cytotoxicity A1-IgG1 37 ± 8 A1-mcMMAF 34 ± 1 A1-IgG4 9 ± 5

EXAMPLE 6 Binding Affinity

Surface plasmon resonance (SPR) analysis is performed utilizing theBIAcore® to determine the affinity constants for A1-IgG1 and A1-IgG4binding to either human or cynomolgus 5T4 at pH 6.0 and pH 7.4. BIAcore®technology utilizes changes in the refractive index at the surface layerupon binding of the huA1 antibody variants to the human 5T4 proteinimmobilized on the surface layer. Binding is detected by SPR of laserlight refracting from the surface. Analysis of the signal kineticson-rate and off-rate allows the discrimination between non-specific andspecific interactions. The 5T4 proteins used for this analysis consistedof the human or cynomolgus 5T4 ectodomain fused to the human IgG1-Fcdomain and low densities (45.1 and 45.4 RU for human and cynomolgusrespectively) are immobilized onto a CM5 chip to accurately measureaffinity constants.

The measurement of specific binding to the 5T4 ectodomain is attained bysubtracting binding to a reference surface that had only human IgG1-Fcprotein immobilized onto the CM5 chip at the same density to that on the5T4-Fc surfaces. Next, various concentrations of A1, A1-IgG4, or A3antibodies in either HBS-EP pH 7.4 or MES-EP pH 6.0 buffer are injectedover the surface. The surface is regenerated two times with Glycine pH1.7+0.05% Surfactant P20 (GE Healthcare, BR-1000-54) between injectioncycles.

Results show that the A1 has a slightly higher affinity for human 5T4using the low-density 5T4 surface at both pH 6.0 and pH 7.4 relative toA1-IgG4 (1.5-fold and 1.2-fold respectively, Table 13). Additionally, A1exhibited slightly better binding to cynomolgus 5T4 at both pH 6.0 andpH 7.4 compared to A1-IgG4 (1.7-fold and 1.2-fold respectively) and bothA1 and A1-IgG4 bound human 5T4, 3-4 fold better than cynomolgus 5T4(Table 12).

TABLE 13 Antibody Antigen pH ka (1/Ms) kd (1/s) KD (nM) A1-IgG1 hu5T46.0 4.31E+05 4.59E−04 1.06 A1-IgG4 hu5T4 6.0 6.26E+05 8.93E−04 1.43A1-IgG1 cyno5T4 6.0 2.33E+05 6.41E−04 2.76 A1-IgG4 cyno5T4 6.0 2.02E+059.50E−04 4.70 A1-IgG1 hu5T4 7.4 2.75E+05 1.32E−04 0.48 A1-IgG4 hu5T4 7.43.28E+05 1.72E−04 0.52 A1-IgG1 cyno5T4 7.4 1.51E+05 2.73E−04 1.80A1-IgG4 cyno5T4 7.4 1.81E+05 3.82E−04 2.11

Comparing the A1 and A3 antibodies, it is apparent that the A1 antibodybinds human and cynomolgus 5T4 better at pH 7.4 relative to pH 6.0 whilethe A3 antibody exhibits enhanced binding at pH 6.0 compared to pH 7.4,Table 14.

TABLE 14 Antibody Antigen pH ka (1/Ms)on kd (1/s)off KD (nM) A1 hu5T46.0 4.31E+05 4.59E−04 1.06 A3 hu5T4 6.0 3.51E+05 4.17E−05 0.12 A1cyno5T4 6.0 2.33E+05 6.41E−04 2.76 A3 cyno5T4 6.0 4.58E+05 1.87E−04 0.41A1 hu5T4 7.4 2.75E+05 1.32E−04 0.48 A3 hu5T4 7.4 1.79E+05 3.06E−05 0.17A1 cyno5T4 7.4 1.51E+05 2.73E−04 1.80 A3 cyno5T4 1.98E+05 1.62E−04 0.821.98E+05

EXAMPLE 7 Epitope Mapping Using 5T4 Chimeras

To identify the epitopes to which each of the A1 and A3 antibodies bind,an enzyme linked immunosorbent assay (ELISA) is performed using (1) 5T4ectodomain Fc construct and (2) human/mouse 5T4 chimera constructstransiently expressed in COS-1 cells. The ectodomain includes theamino-terminal region, two leucine-rich repeats, and the interveninghydrophilic region. Mouse and rat 5T4 ectodomains contain a 6 amino aciddirect repeat within their hydrophilic region.

Fusion proteins containing a 5T4 ectodomain and a Fc constant regionfrom human IgG1 are prepared using human 5T4 (amino acids 1-355), mouse5T4 (amino acids 1-361), rat 5T4 (amino acids 1-361), cynomologus monkey5T4 (amino acids 1-355), chimpanzee 5T4 (amino acids 1-355), andblack-tailed marmoset (amino acids 1-355). The binding results withhuman/mouse 5T4 chimera constructs are summarized in Table 14, whichindicates specific binding, partial binding, or lack of binding, by theA1 and A3 antibodies.

Table 15 refers to binding ability of the antibodies to the varioushuman/mouse chimeras and the nomenclature is designated by mouse 5T4content. When no binding is observed, this indicates where the antibodybinds human 5T4 since these antibodies do not bind mouse 5T4. Forexample, the A3 antibody has the most N-terminal binding epitope(between 83-163) and this is shown by lack of binding to the 5T4 chimerathat has residues 83-163 replaced by mouse 5T4, hence A3 can no longerbind. Based upon these results, it is determined that humanized A1antibody has a first contact with human 5T4 between amino acid residues173 and 252 and a second contact with human 5T4 between amino acidresidues 276 and 355. The A3 antibody binds the first leucine-richrepeat region of human 5T4 between amino acid residues 83 through 163.The number of amino acid residues corresponds to the human 5T4 antigenamino acid sequence of SEQ ID NO: 11.

TABLE 15 Antibody 5T4 Antigen Construct A1 A3 Human/mouse 83-163 + −Human/mouse 173-361 − + Human/mouse 173-258 +/− + Human/mouse 282-361+/− +

EXAMPLE 8 Comparison of A1-mcMMAF ADC with A1-IgG4-CM ADC

A1-mcMMAF is compared to A1-IgG4-AcBut calicheamicin (A1-IGG4-CM) forboth safety and efficacy. A1-4-CM is comprised of the A1-IgG4 antibodyconjugated with the linker, AcBut[-(4′ acetylphenoxy)butanoic acid], toa calicheamicin payload. The calicheamicins are potent antitumor agentsof a class of enediyne antibiotics derived from the bacteriumMicromonospora echinospora.

The cell binding activity of A1 Ab, A1-IgG4 Ab, A1-mcMMAF ADC andA1-IgG4-CM ADC are compared using several 5T4 positive cell lines (seeExample 2, Table 5). The data indicates that similar binding is observedwith the A1 and A1-IgG4 antibodies as well as the A1-mcMMAF ADC, all ofwhich have a higher mean fluorescent intensity than A1-IgG4-CM for allthe 5T4 positive cell lines tested.

A1-mcMMAF and A1-IgG4-CM are tested side-by-side in the MDAMB435/5T4subcutaneous xenograft model. Both ADCs are given iv (Q4d×2) when thetumors reach approximately 200 mm² in size. The anti-tumor activity ofA1-IgG4-CM at a dose of 3 mg/kg is similar to the anti-tumor activity ofA1-mcMMAF administered at dose of 10 mg/kg (Table 16). Based upon theseresults, the anti-tumor activity of A1-IgG4-CM is approximately 3.3 foldmore potent than A1-mcMMAF.

TABLE 16 Tumor volume (mm³, x ± sem) Dose (mg/kg) Compound Q4dx4 Day 0Day 7 Day 21 Day 31 Day 45 Vehicle  0 123 ± 8  195 ± 36 402 ± 56 635 ±111 1309 ± 332 A1-  3 124 ± 11 121 ± 8  166 ± 29 227 ± 42  361 ± 89mcMMAF A1- 10 123 ± 14  76 ± 11  0 ± 0 3 ± 3  2 ± 1 mcMMAF A1-IGG4-  3121 ± 12 140 ± 15  32 ± 10 24 ± 10  26 ± 15 CM

It could be expected that the 3.3 fold enhanced potency of A1-IgG4-CMover that of A1-mcMMAF would translate into a 3.3 fold enhanced safetymargin of A1-mcMMAF over that of A1-IgG4-CM in an animal toxicity study.However, when the safety profile of A1-IgG4-CM in cynomolgus macques isreviewed, it is determined that A1-IgG4-CM is at least 100 fold moretoxic than A1-mcMMAF in the cynomolgus macque. When A1-IgG4-CM isadministered at 0.032, 0.095 and 0.32 mg Ab/kg/cycle (2, 6, 20 μgcalicheamicin/kg/cycle) to male (n=3) and female (n=3) cynomolgusmacques, toxicity is observed at each dose level. After 2 cycles (2doses), 4 out of 6 animals in the 0.095 treatment group are eithereuthanized or found dead. On the other hand, no deaths are observed atdosages up to 10 mg/kg with A1-mcMMAF (247 pg mcMMAF/kg/cycle), after 2cycles (2 doses), over the same 4 week time period. In summary, the 10mg/kg dosage group of A1-mcMMAF is safe while the 0.096 mg/kg dosagegroup of A1-IgG4-CM is deemed toxic when both are administered twice tocynomolgus macques in a 4 week observation period.

Unexpectedly, these results demonstrate a 105 fold (10/0.095=105) safetymargin of A1-mcMMAF over that of A1-IgG4-CM, rather than the expected3.3 fold safety margin based on the relative anti-tumor potency of eachADC. This data reveals the unpredictable nature of antibody-drugconjugates that utilize antibodies to the same antigen target but areconjugated to a different drug payload.

EXAMPLE 9 A1-mcMMAF Mouse PK/PD Modeling and Clinical Dose Predictions

PK/PD modeling has been used to quantify the tumor response of A1-mcMMAFin mouse xenograft studies, in order to determine efficaciousconcentration across cell lines. The transit compartment tumor killPK/PD model used was previously described by Simeoni et al. (Simeoni etal, Cancer Res, 64:1094, (2004). The model has been modified to accountfor linear, exponential and logistic growth of tumor, and saturativekilling by the drug. PK/PD model parameters include:

-   -   k_(g ex) exponential growth    -   k_(g) logistic growth    -   w₀ initial tumor volume    -   tau transduction rate    -   k_(max) maximum kill rate    -   kC₅₀ concentration at half max kill rate

The PK/PD modeling results are used to calculate the Tumor StaticConcentration (TSC, Equation 1). This is the drug concentration wheretumor growth is equal to tumor death rates and tumor volume remainsunchanged. TSC can be defined as the minimal concentration required forefficacy. TSC is used to give guidance on clinical dose selection, withconcentrations of >TSC required for efficacy in the clinic.

For A1-mcMMAF, mouse PK was determined in a separate study (3mg/kg IV,female athymic nu/nu mice). Mouse xenograft studies were completed using3 different 5T4 cell lines with A1-mcMMAF administered at dose levelsbetween 1 and 30 mg/kg every 4 days: cell line MDAMB435/5T4 (dosed at 1,3, 10, and 30 mg/kg), cell line H1975 (dosed at 1, 3, and 10 mg/kg) andcell line 37622A (dosed at 1 and 10 mg/kg). PK/PD modeling was performedas described and TSCs are reported in Table 17.

Mouse PK/PD parameters for each xenograft cell line were combined withpredicted human PK of A1-mcMMAF to simulate doses required for efficacyin the clinic. Using this methodology, A1-mcMMAF has a predictedminimally efficacious clinical dose of about 0.22 to about 2.3 mg/kg Q3weeks [every three weeks] (Table 17).

In an embodiment of the present invention, dose ranges can be in therange from about 0.18 mg/kg to about 2.7 mg/kg, from about 0.22 mg/kg toabout 2.6 mg/kg, from about 0.27 mg/kg to about 2.5 mg/kg, from about0.32 mg/kg to about 2.3 mg/kg, from about 0.37 mg/kg to about 2.15mg/kg, from about 0.42 mg/kg to about 2.10 mg/kg, from about 0.47 mg/kgto about 2.05 mg/kg, from about 0.52 mg/kg to about 2.00 mg/kg, fromabout 0.57 mg/kg to about 1.95 mg/kg, from about 0.62 mg/kg to about1.90 mg/kg, from about 0.67 mg/kg to about 1.85 mg/kg, from about 0.72mg/kg to about 1.80 mg/kg, from about 0.82 mg/kg to about 1.70 mg/kg,from about 0.92 mg/kg to about 1.60 mg/kg, from about 1.02 mg/kg toabout 1.50 mg/kg, from about 1.12 mg/kg to about 1.40 mg/kg, or fromabout 1.20 mg/kg to about 1.30 mg/kg, with dosing at Q3 weeks.Preferably, dose ranges can be in the range from about 0.22 mg/kg toabout 2.3 mg/kg.

$\begin{matrix}{{{Equation}\mspace{14mu} 1}} & \; \\\begin{matrix}{{{{If}\mspace{14mu} \frac{k_{gEx}}{k_{g}}w_{0}} \leq 1},} & {{TSC} = \frac{k_{gEx} \cdot k_{C\; 50}}{k_{\max} - k_{gEx}}}\end{matrix} & 1.1 \\\begin{matrix}{{{{If}\mspace{14mu} \frac{k_{gEx}}{k_{g}}w_{0}} > 1},} & {{TSC} = \frac{k_{g} \cdot k_{C\; 50}}{{w_{0} \cdot k_{\max}} - k_{g}}}\end{matrix} & 1.2\end{matrix}$

TABLE 17 TSC Predicted Stasis Dose [80% confidence] [80% confidence]Cell Line (ug/ml) (mg/kg Q3 weeks) MDAMB435/5T4 1.1 [0.9, 1.4]  0.22[0.18, 0.28] 37622A 5.1 [2.1, 9.9] 1.1 [0.6, 2.0] H1975 11.6 [9.6, 14.1]2.3 [2.0, 2.7]

SEQUENCE LISTING

Humanized A1 human IgG1 heavy chain  SEQ ID NO: 1EVQLVESGGGLVQPGGSLRLSCAASGYTFTNFGMNWVRQAPGKGLEWVAWINTNTGEPRYAEEFKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDWDGAYFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Humanized A1 human Kappa light chain:  SEQ ID NO: 2DIQMTQSPSSLSASVGDRVTITCKASQSVSNDVAWYQQKPGKAPKLLIYFATNRYTGVPSRFSGSGYGTDFTLTISSLQPEDFATYYCQQDYSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC A1-VH  SEQ ID NO: 3 EVQLVESGGGLVQPGGSLRLSCAASGYTFTNFGMNWVRQAPGKGLEWVAWINTNTGEPRYAEEFKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDW DGAYFFDYWGQGTLVTVSS A1-VL  SEQ ID NO: 4 DIQMTQSPSSLSASVGDRVTITCKASQSVSNDVAWYQQKPGKAPKLLIYFATNRYTGVPSRFSGSGYGTDFTLTISSLQPEDFATYYCQQDYSSPWTFGQ GTKVEIK  A1-HC CDR1 SEQ ID NO: 5 NFGMN  A1-HC CDR2  SEQ ID NO: 6 WINTNTGEPRYAEEFKG A1-HC CDR3  SEQ ID NO: 7 DWDGAYFFDY  A1-LC-CDR1  SEQ ID NO: 8KASQSVSNDVA  A1-LC-CDR2  SEQ ID NO: 9 FATNRYT  A1-LC-CDR3  SEQ ID NO: 10QQDYSSPWT  Human 5T4 antigen  SEQ ID NO: 11MPGGCSRGPAAGDGRLRLARLALVLLGWVSSSSPTSSASS FSSSAPFLASAVSAQPPLPDQCPALCECSEAARTVKCVNR NLTEVPTDLPAYVRNLFLTGNQLAVLPAGAFARRPPLAEL AALNLSGSRLDEVRAGAFEHLPSLRQLDLSHNPLADLSPF AFSGSNASVSAPSPLVELILNHIVPPEDERQNRSFEGMVV AALLAGRALQGLRRLELASNHFLYLPRDVLAQLPSLRHLD LSNNSLVSLTYVSFRNLTHLESLHLEDNALKVLHNGTLAE LQGLPHIRVFLDNNPWVCDCHMADMVTWLKETEVVQGKDR LTCAYPEKMRNRVLLELNSADLDCDPILPPSLQTSYVFLG IVLALIGAIFLLVLYLNRKGIKKWMHNIRDACRDHMEGYH  YRYEINADPRLTNLSSNSDV Humanized A1 human IgG4m heavy chain:  SEQ ID NO: 12EVQLVESGGGLVQPGGSLRLSCAASGYTFTNFGMNWVRQAPGKGLEWVAWINTNTGEPRYAEEFKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDWDGAYFFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK A1 human IgG4m VH (A1-IGG4-VH)  SEQ ID NO: 13EVQLVESGGGLVQPGGSLRLSCAASGYTFTNFGMNWVRQAPGKGLEWVAWINTNTGEPRYAEEFKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDW DGAYFFDYWGQGTLVTVSS A1-IgG4-VH-CDR1  SEQ ID NO: 14 NFGMN Chimeric A3 heavy chain (muA3-huIgG1)  SEQ ID NO: 15EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSQSMLYLQMNNLKTEDTAMYYCVRQWDYDVRAMNYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK  Chimeric A3 VH SEQ ID NO: 16 EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSQSMLYLQMNNLKTEDTAMYYCVRQWDYDVRAMNYWGQGTLVTVSS  Chimeric A3 VH-CDR1  SEQ ID NO: 17 TYAMN Chimeric A3 VH-CDR2  SEQ ID NO: 18 RIRSKSNNYATYYADSVKD Chimeric A3 VH-CDR3  SEQ ID NO: 19 QWDYDVRAMNY Chimeric A3 light chain (muA3-huKappa)  SEQ ID NO: 20DIVMTQSHIFMSTSVGDRVSITCKASQDVDTAVAWYQQKPGQSPKLLIYWASTRLTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC Chimeric A3 VL  SEQ ID NO: 21DIVMTQSHIFMSTSVGDRVSITCKASQDVDTAVAWYQQKPGQSPKLLIYWASTRLTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYPYTFGQ GTKLEIK Chimeric A3 VL-CDR1  SEQ ID NO: 22 KASQDVDTAVA  Chimeric A3 VL-CDR2 SEQ ID NO: 23 WASTRLT  Chimeric A3 VL-CDR3  SEQ ID NO: 24 QQYSSYPYT Humanized A3 human IgG1 heavy chain:  SEQ ID NO: 25EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDAKNSLYLQMNSLRAEDTAVYYCVRQWDYDVRAMNYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK  Humanized A3 VH: SEQ ID NO: 26 EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDAKNSLYLQMNSLRAEDTAVYYCVRQWDYDVRAMNYWGQGTLVTVSS  Humanized A3 VH-CDR1:  SEQ ID NO: 27 TYAMN Humanized A3 VH-CDR2:  SEQ ID NO: 28 RIRSKSNNYATYYADSVKD Humanized A3 VH-CDR3:  SEQ ID NO: 29 QWDYDVRAMNY Humanized A3 human Kappa light chain:  SEQ ID NO: 30DIQMTQSPSSLSASVGDRVTITCKASQDVDTAVAWYQQKPGKAPKLLIYWASTRLTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC Humanized A3 VL:  SEQ ID NO: 31DIQMTQSPSSLSASVGDRVTITCKASQDVDTAVAWYQQKPGKAPKLLIYWASTRLTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSYPYTFGQ GTKLEIK Humanized A3 VL-CDR1:  SEQ ID NO: 32 KASQDVDTAVA  Humanized A3 VL-CDR2: SEQ ID NO: 33 WASTRLT  Humanized A3 VL-CDR3:  SEQ ID NO: 34 QQYSSYPYT 

We Claim:
 1. An antibody-drug conjugate of the formula:Ab-(LU-D)p or a pharmaceutically acceptable salt thereof wherein; (a) Abis an anti-5T4 antibody or antigen binding portion thereof, comprising:(i) a VH CDR1 region as shown in SEQ ID NO: 5, (ii) a VH CDR2 region asshown in SEQ ID NO: 6, (iii) a VH CDR3 region as shown in SEQ ID NO: 7;(iv) a VL CDR1 region as shown in SEQ ID NO: 8, (v) a VL CDR2 region asshown in SEQ ID NO: 9, and (vi) a VL CDR3 region as shown in SEQ ID NO:10, (b) LU is a linker unit selected from the group consisting ofmaleimidocaproyl and maleim idocaproyl-Val-Cit, (c) p is an integer fromabout 1 to about 8, and (d) D is a Drug unit selected from the groupconsisting of MMAE, MMAD, and MMAF.
 2. The antibody-drug conjugate ofclaim 1 wherein said anti-5T4 antibody or antigen binding portionthereof, comprises the VH region of SEQ ID NO: 3 and the VL region ofSEQ ID NO:
 4. 3. The antibody-drug conjugate of claim 2 wherein saidanti-5T4 antibody or antigen binding portion thereof, consists of aheavy chain having SEQ ID NO:1 and a light chain having SEQ ID NO:
 2. 4.The antibody-drug conjugate of claim 3, wherein said antibody or antigenbinding portion thereof, recognizes an epitope on human 5T4 antigen,wherein said epitope comprises amino acid residues 173-258 and 282-361of the amino acid sequence of SEQ ID NO:11.
 5. The antibody-drugconjugate of claim 1 wherein: (a) said anti-5T4 antibody consists of aheavy chain having SEQ ID NO:1 and a light chain having SEQ ID NO: 2,(b) said LU is maleimidocaproyl, (c) said Drug is MMAF, and (d) p is 4.6. A pharmaceutical composition comprising the antibody-drug conjugateof claim 1 and a pharmaceutically acceptable carrier.
 7. A method oftreating 5T4-positive cancer in a patient in need thereof, comprisingadministering to said patient the antibody-drug conjugate according toclaim
 1. 8. The method of claim 7 wherein said cancer is selected fromthe group consisting of colorectal, breast, pancreatic, and non-smallcell lung carcinomas.